Gene therapy is generally understood to refer to techniques designed to deliver nucleic acids, including antisense DNA and RNA, ribozymes, viral genome fragments and functionally active therapeutic genes into targeted cells (Culver, 1994, Gene Therapy: A Handbook for Physicians, Mary Ann Liebert, Inc., New York, N.Y.). Such nucleic acids can themselves be therapeutic, as for example antisense DNAs that inhibit mRNA translation, or they can encode, for example, therapeutic proteins that promote, inhibit, augment, or replace cellular functions.
A serious shortcoming of current gene therapy strategies, including both ex vivo and in vivo gene therapy methods, is the inability of present vector and delivery system combinations to deliver nucleic acids efficiently into the interior of cells of a targeted population. In December, 1995, the U.S. National Institutes of Health issued a "Report and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy" (Orkin et al., 1995, National Institutes of Health, Bethesda, Md.). In this Report, it was recognized that the development of gene therapy approaches to disease treatment was being inhibited, in part, by a dearth of effective gene transfer vectors. The Report recognized a need for further research applied to improving vectors for gene delivery.
Virus vectors are generally regarded as the most efficient nucleic acid delivery vectors. Recombinant replication-defective virus vectors have been used to transduce (i.e., infect or transfect) animal cells both in vitro and in vivo. Such vectors have included retrovirus, adenovirus, adeno-associated virus vectors, and herpesvirus vectors. Although they are highly efficient for gene transfer, a major disadvantage associated with the use of virus vectors is the inability of many virus vectors to infect non-dividing cells. Another serious problem associated with the use of virus gene vectors is the potential for such vectors to induce an immune response in a patient to whom they are administered. Such an immune response limits the effectiveness of the virus vector, since the patient's immune system rapidly clears the vector upon repeated or sustained administration of the vector. Furthermore, insertion of a gene into the genome of a cell by a virus vector can induce undesirable mutations in the cell. Other problems associated with virus gene vectors include inability to appropriately regulate gene expression over time in transfected cells, toxicity and other side effects caused by delivery of virus vectors to human tissues (e.g. liver damage and myocarditis), and potential production and transmission to other humans of harmful virus particles.
Furthermore, virus gene vectors, as used in prior art methods, have the drawback that they often cannot be delivered to a selected tissue in a specific, localized manner. Instead, many prior art methods of administering virus vectors result in vector being dispersed systemically or to tissues which adjoin, or are in fluid communication with, the desired target tissue. The inability of such methods to localize virus vector reduces the utility of the methods, because non-localized virus vector can transfect unintended tissues, elicit immune responses, be rapidly excreted from the body, or otherwise suffer diminished transfection ability. A significant need exists for methods of delivering virus vectors in a localized manner.
Virus vectors are able, to a limited degree, to deliver proteins and other therapeutic molecules to the cells which the virus vectors transfect. Such proteins and other therapeutic molecules can be incorporated passively and non-specifically into virus vector particles. Alternatively, as is known in the art, certain virus vectors specifically incorporate fusion proteins comprising a protein having a polypeptide viral packaging signal fused therewith.
Even though virus vectors have been widely used in experimental gene therapy protocols and human studies (Feldman et al., 1997, Cardiovasc. Res. 35:391-404; Roth et al., 1997, J. Natl. Cancer Inst. 89:21-39), none of these vectors has proven efficacious for virus vector-mediated gene therapy. It has been hypothesized that the shortcomings of adenovirus vectors has been due, at least in part, to limited transgene expression resulting from the immune response of the host individual and to cytotoxic effects which the vectors have exhibited toward organs of the host individual (Smith et al., 1996, Gene Ther. 3:190-200; Tripathy et al., 1996, Nat. Med. 2:545-549; Nabel et al., 1995, Gene Ther. Cardiovasc. Dis. 91:541-548). Others working in the field have concentrated their efforts on mutating adenovirus vectors to render them relatively less immunogenic and toxic.
In addition to the low efficiency of virus vector uptake exhibited by most cell types and low levels of expression of the gene constructs delivered by virus vectors, many targeted cell populations are found in such low numbers in the body that the efficiency of transfection of these specific cell types is even further diminished. A critical need remains for gene therapy methods which can efficiently deliver virus vectors to targeted cell populations. Others working in the field have concentrated on attempting to specifically target adenovirus vectors to a particular cell type, for example by attaching a specialized receptor ligand to the vectors (Tzimagiorgis et al., 1996, Nucl. Acids 24:3476-3477).
A virus vector useful for gene delivery must be delivered to its target cells in a form in which the biochemical components of the virus retain their function. That is, the virus vector must retain the capacity to bind to target cells, to transfer a nucleic acid carried by the vector into the interior of the cell, and, in some circumstances, to catalyze chemical reactions involving that nucleic acid within the cell (e.g. reverse transcription, integration into the host cell genome, or promoting transcription of gene elements on the nucleic acid). Thus, it is important that the delivery vehicle by means of which the virus vector is administered to a patient not subject the vector to chemically harsh or biochemically inactivating conditions. Thus, many matrices are not compatible for contacting with virus vectors. Ideally, a matrix in or on which a virus vector is disposed should be biodegradable, and in a form which is amenable to use in relevant surgical and therapeutic interventions. Further complicating matters, the following physiological phenomena are some of those which can inhibit administration of a virus vector to an animal tissue.
Inability of the virus vector to interact specifically with cells of the desired tissue attributable to proteolytic degradation of one or more components of the virus vector by an enzyme in the animal. PA1 Complexing of animal proteins or other molecules with one or more components of the virus vector, with the result that the virus vector is unable to interact specifically with cells of the desired tissue. PA1 Sequestration of the virus vector in undesired tissues or organs of the animal (e.g. removal of virus vectors from the bloodstream by the liver). PA1 Complications (e.g. immune reactions, inappropriate transfection, rapid clearance of vector from the subject, etc.) arising from non-localized delivery of the virus vector. PA1 Inability of the virus vector to cross a physical barrier (e.g. the blood-brain barrier or peritoneal membranes) which separates the desired tissue from the site of administration of the virus vector. PA1 Induction of an immune response in the animal which results in production in the animal of cells and proteins (e.g. antibodies) which inactivate the virus vector. PA1 Relatively short duration of the period during which the virus vector contacts the desired soft tissue, either due to the immune response described above or due to rapid interaction of all available virus vector particles with the desired tissue.
A desirable virus vector will permit administration that is not significantly inhibited by these phenomena.
Hydrogels are synthetic polymer or biopolymer matrices that are highly hydrated (e.g. at least 50%, by weight, of the hydrogel comprises water). Despite the high degree of hydration of hydrogels, an important characteristic of hydrogels is that they are structurally stable. Commonly used hydrogels include those composed of synthetic components, such as polyacrylamides or poloxamers. Other hydrogels which have been used are composed of a naturally occurring polymer, such as collagen. For example, freeze-dried collagen matrices have been used to deliver plasmid DNA to bone tissue in order to encourage bone regeneration (Fang et al., 1996, Proc. Natl. Acad. Sci. USA 93:5753-5758). However, hydrated collagen gels lack structural integrity. Alginate, a polysaccharide derived from algae, forms an insoluble aggregate in the presence of calcium, and such calcium alginates are known to be effective immobilizing agent. Alginates have been used in combination with polyamines to deliver rotavirus vaccines to gastrointestinal tissues (Moser et al., 1996, Vaccine 14:1235-1238).
Other investigators have incorporated biomaterials, such as enzymes and living cells, into hydrogel matrices (e.g. U.S. Pat. No. 4,004,979, U.S. Pat. No. 4,452,892, U.S. Pat. No. 4,647,536, and U.S. Pat. No. 5,648,252). In some instances, these investigators have demonstrated that the hydrogel-incorporated cells survived or that the hydrogel-incorporated enzymes retained their enzymatic activity. One group of investigators (U.S. Pat. No. 5,529,777) incorporated virus particles into hydrogel and demonstrated the usefulness of hydrogel-incorporated virus particles as a vaccine. However, these investigators do not describe a composition or method for maintaining the infectivity of the virus particles. Of course, such infectivity would be contrary to use of the composition as a vaccine.
The biocompatibility of many hydrogel compositions, combined with their structural strength and biodegradability, recommends the use of hydrogels as virus vector delivery vehicles. Unfortunately, however, many of the synthetic components commonly used to form hydrogels are incompatible with maintaining virus infectivity.
A critical need remains for hydrogel compositions in which virus vectors can be maintained in an infective state, particularly in a localized manner. The hydrogel compositions described herein satisfy this need.